The expression of human telomerase reverse transcriptase (hTERT) is present in most malignant cells (.85%) but is undetectable in most normal somatic cells. Visualization of hTERT expression using radionuclide targeting can provide important diagnostic information in malignant tumors. The overall aim of this study was to evaluate whether 99m Tc-radiolabeled antisense oligonucleotide (ASON) targeting hTERT messenger RNA (mRNA) can be used for imaging of hTERT expression in vivo. Methods: One 18-mer antisense or sense uniformly phosphorothioate-modified oligonucleotide targeting hTERT mRNA was radiolabeled with 99m Tc through the bifunctional chelator N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (S-acetyl NHS-MAG3). Then the radiolabeled probe was characterized in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to assay the mRNA level after proliferating cells had been incubated with the antisense and sense probes. 99m Tc-MAG3-ASON or 99m Tc-MAG3-SON was injected intravenously in mammary tumor-bearing BALB/c nude mice. Biodistribution and in vivo imaging was performed periodically. All data were analyzed by statistical software. Results: The labeling efficiencies of 99m Tc-MAG3-ASON/SON reached 76% 6 5% (n 5 5) within 15-30 min at room temperature, the specific activity was up to 1,850 kBq/mg, and the radiochemical purity was .96% after purification. 99m Tc-MAG3-ASON showed complete stability at room temperature and in fresh 37°C human serum. In comparison with 99m Tc-MAG3-SON, 99m Tc-MAG3-ASON preserved the capacity to bind living hTERT-expressing cells specifically and to inhibit the expression of hTERT mRNA significantly as well as ASON. In nude mice bearing hTERT-expressing MCF-7 xenografts, tumor radioactivity uptake of 99m Tc-MAG3-ASON after injection was significantly higher than that of 99m Tc-MAG3-SON after injection (P , 0.05). The hTERT-expressing xenografts were clearly imaged at 4-8 h noninvasively after injection of 99m Tc-MAG3-ASON, whereas the xenografts were not imaged at any time after injection of 99m Tc-MAG3-SON. Conclusion: This in vivo study provides evidence that 99m Tc-MAG3-ASON targeting hTERT mRNA can be used as a potential candidate for visualization of hTERT expression in carcinomas.

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Small-interference RNAs (siRNAs) are short, double-strand RNA molecules that target specific messenger RNAs for degradation via the process termed RNA interference. The efficacy of RNA interference depends greatly on effective delivery of siRNA, which calls for noninvasive methods for tracing siRNA in vivo. The purpose of this work was to develop a novel 99m Tc-radiolabeled method to visualize siRNA targeting of a tumor biomarker of human telomerase reverse transcriptase (hTERT) in HepG2 tumor xenografts. Methods: After conjugation with S-acetyl N-hydroxysuccinimide-mercaptoacetyltriglycine (NHS-MAG3), antisense RNA with 29-O-methyl modification was annealed with sense strand to form a duplex and then radiolabeled with 99m Tc. 99m Tc-siRNAs were tested for stability in serum by measurement of radiochemical purity and for inhibitory activity by reverse-transcriptase polymerase chain reaction and Western blotting. In vitro cellular uptake was evaluated in HepG2 cells. Biodistribution studies and static imaging were performed in HepG2 tumor-bearing mice. Results: Radiochemical purity remained highly stable in saline and fresh human serum at room temperature and 37°C. Radiolabeled siRNA demonstrated strong inhibitory effects similar to those of unlabeled siRNA on both hTERT messenger RNA and protein in vitro. 99m Tc-hTERT siRNA showed more uptake than did control siRNA in HepG2 cells after 1 h of incubation. After administration in HepG2 tumor-bearing mice, 99m Tc-hTERT siRNA had significantly higher accumulation in tumors and a higher tumor-to-blood ratio than did control siRNA (P , 0.05). Scintigraphy of 99m Tc-hTERT siRNA showed clear tumor images at 0.5, 1, 3, and 6 h after injection. In contrast, 99m Tc-control siRNA failed to visualize the tumor. Ratios of uptake in tumor to uptake in contralateral region of hTERT-targeted siRNA were significantly higher than those of control siRNA (P , 0.05) at each time point. Conclusion: The 99m Tc radiolabeling method with NHS-MAG3 chelator can be used successfully in siRNA radiolabeling, allowing for the noninvasive visualization of siRNA delivery in vivo.Key Words: 99m Tc; small-interference RNA (siRNA); delivery; human telomerase reverse transcriptase (hTERT); molecular imaging J Nucl Med 2010; 51:978-986

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The results suggest that radioiodination of RRL peptide is feasible and that the labeled compound is stable in human blood serum. The (131)I-labeled RRL peptide shows high tumor uptake and good tumor-to-organ ratios that allow noninvasive visualization of tumors. The (131)I-labeled compound is valuable to detect tumors as molecular probe.